CFTR (cystic fibrosis transmembrane conductance regulator), the product of the gene mutated in CF, is an ABC protein that forms a Cl- ion channel. Its opening and closing are controlled by cycles of ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBDs) whose function, in turn, is regulated by protein-kinase-A-mediated phosphorylation of multiple serines in CFTR's regulatory domain. The goal of the parent grant is to understand, in molecular detail, the structure and mechanisms of function of the NBDs, the interactions between them, and the mechanisms by which they are regulated. Towards that goal the parent grant exploits electrophysiological, biochemical, molecular biological, and biophysical methods (including structural modeling and, hopefully, eventually crystallography) to characterize the function of WT and mutant CFTR protein, over a wide range of conditions. This FIRCA proposal seeks funds for additional detailed measurements and analysis of microscopic gating kinetics to be carried out primarily by Dr. Csanady at the foreign site (Semmelweis University Medical School, in Budapest, Hungary). This additional proposed work will greatly extend and enhance the analytical power of the parent NIH grant R0l DK51767, by developing and testing appropriate powerful Markov kinetic schemes that will explain all data on the function of WT and mutant CFTR channels, whether obtained at the parent or foreign site (or indeed all reliable data obtained by any investigator anywhere). The ongoing close, productive, working relationship developed between the P1 and Dr. Csanady over the past approximately 5 years (during the latter's PhD work at Rockefeller) ensures that the proposed collaborative work will succeed. The U.S. group will provide molecular biological and biochemical support: specifically, we will supply Dr. Csanady with purified PKA catalytic subunit, cDNAs of WT and mutant CFTRs, and provide overall biochemical and electrophysiological characterizations of those and other CFTR constructs. At the foreign site, Dr. Csanady will express the cDNAs in oocytes, make detailed measurements of CFTR channel gating kinetics in excised patches under conditions designed to emphasize diagnostic power, and analyze those kinetics using his custom-developed software that harnesses maximum likelihood optimizations, with the aim of deriving the most appropriate Markov schemes to fully describe, quantitatively, the processes that control opening and closing of the CFTR channel pore. The two groups will closely interact to maximize the progress of both projects.